The receptors that mediate chemotaxis by Escherichia coli are the best characterized members of a large family of bacterial chemoreceptors and a larger family of prokaryotic and eukaryotic sensory receptors. This proposal focuses on sensory transduction by these chemoreceptors, with emphasis on transmembrane and intermolecular signaling. The proposed work is a combination of biochemical, biophysical, genetic and structural approaches, concentrating in many projects on the chemoreceptor Trg. In the current funding period the PI created, characterized and utilized a collection of Trg variants with one or two cysteines per polypeptide chain, placed primarily in one or both transmembrane segments. Extensive use will be made of this collection of mutant receptors in experiments designed to address central issues of transmembrane organization and signaling. The principal aims of the proposal are to: (1) determine helical interactions in the transmembrane domain; (2) measure distances between pairs of positions in the transmembrane segments, or between a specific position and the membrane surface; and (3) define the molecular details of the conformational change involved in transmembrane signaling. Several of these studies will include the use of electron paramagnetic resonance spectroscopy and will be done in collaboration with Dr. Wayne Hubbell. In other studies the interaction of chemoreceptor and ligand-occupied binding proteins will be investigated using surface plasmon resonance. The "minority" chemoreceptor Trg is effective in ligand-induced signaling but defective in steady-state activation of the CheA signaling kinase. The basis of this signaling behavior will be investigated by physiological, genetic and biochemical studies with the hope of learning about the little-understood phenomenon of signal amplification in the bacterial chemosensory system. In addition, genetic and biochemical approaches will be used to characterize interactions between the three chemoreceptor domains, and efforts will be directed toward obtaining particles and two-dimensional crystal of receptor-CheW-CheA complexes suitable for structural analysis by electron microscopy.